Lymph is rich in lipids, including lipoproteins, and the return of cholesterol from tissues to the vasculature and ultimately the liver depends not only on the loading of cholesterol onto high-density lipoprotein (HDL) particles within parenchymal tissues but also access of the HDL to the bloodstream. The lymphatic system participates centrally in returning HDL to the bloodstream via the thoracic duct, and it also absorbs lipoproteins directly from the small intestinal villi in the form of chylomicrons. Thus, though it is rarely considered, lymphatic vessels are likely to be key participants in cholesterol homeostasis. The importance of this concept became clearer to us when we observed that mice with primary lymphedema, devoid of dermal lymphatic capillaries, unexpectedly had elevated levels of circulating plasma cholesterol. When we also observed that another mouse strain engineered to have hypercholesterolemia--apolipoprotein E (apoE) null mice--develop impaired lymphatic flow and dendritic cell (DC) migration through afferent lymphatics, we began to explore the possibility that cholesterol metabolism and lymphatic function are two processes that are tightly linked and that each regulates the other. Thus, we were led to test whether statins, drugs widely used to lower cholesterol, could improve lymphatic transport, and we have found that this is the case, surprisingly even in models of localized, secondary lymphedema where a link to cholesterol metabolism has not been previously made. In the blood vasculature, it is known that hypercholesterolemia induces endothelial dysfunction by negatively affecting nitric oxide synthases (NOS) and production of nitric oxide (NO), and NOS activity in blood vessels can be restored by statins. NO preserves vascular endothelial function in part by inhibiting angiotensin II. However, the relevance of these mechanisms to lymphatic vessel function, and ultimately lymphedema, has not been widely considered. Thus, we will herein test the hypothesis that cholesterol regulates lymphatic vessel function, and vice versa, and that it does so through its effects on the production of NO. We predict, and will evaluate experimentally in this proposal, that by counteracting VLDL/LDL-mediated endothelial damage, high-density lipoprotein (HDL) is especially important in maintaining lymphatic collecting vessels in an optimally functional state that permits normal lymphatic transport of molecules and DCs. Based on our preliminary data, we hypothesize that HDL does this by ensuring that NOS activity remains active in collecting vessels, generating appropriate levels of NO that suppress angiotensin II signaling. To test this hypothesis, we will bring together our respective expertise in DC trafficking, cholesterol, and lymphatic biology and physiology to a) Delineate the role of HDL in regulating lymphatic flow and dendritic cell migration (aim 1), b) Evaluate whether alterations in cholesterol homeostasis in mice impair lymphatic function by suppressing nitric oxide synthesis pathways (aim 2), and c) Assess the hypothesis that angiotensin II receptor activity is heightened in mouse models of lymphedema and promotes swelling, elevated plasma cholesterol levels, and adipocyte hypertrophy (aim 3). PUBLIC HEALTH RELEVANCE: Lymphatic diseases have received insufficient attention in the past, such that progress in treating lymphatic disease has been slowed. Our three interactive, but independent, aims will bring together scientists from diverse disciplines to address significant aspects of the regulation of lymphatic function. Several promising tools that may ultimately lead to valuable insight into how lymphatic disorders could be treated will be explored in this proposal.